New strategies are needed to block malaria transmission and eradicate the spread of the disease. None of the commonly used antimalarials are effective against Plasmodium gametocytes, the parasite stage responsible for transmission, and consequently patients can remain infectious for weeks after symptoms have resolved. Our recent screen of > 6000 bioactive molecules, including 400 malaria box compounds identified 7 with < 50nM IC50 against stage III-V gametocytes. One of these, Torin 2 had an IC50 of 8 nM against P. falciparum strain 3D7, which is >1000 times lower than its activity against the mammalian HepG2 cell line. Torin 2 was also equally effective against 2 additional P. falciparum strains, HB3 and Dd2, with distinct geographic origins and drug resistance profiles. Importantly, two 4mg/kg doses completely blocked oocyst formation using the mouse P. berghei model to test in vivo transmission blocking activity. Early structure activity relationship (SAR) analysis done in collaboration with investigators at the National Center for Advancing Translational Research (NCATS) allowed the production of a Torin 2 resin that lead to the identification of Torin 2 interacting proteins. The goals of this proposal are to extend our analysis of Torin 2 by: Aim 1: Define the stage specificity and timing of Torin 2 inhibition from intraerythrocytic development to sporozoite formation to inform both drug delivery strategies and mechanistic studies. Aim 2: Evaluate the in vivo activity of novel Torin 2 analogs using the rodent malaria model: Aim 3: Evaluate the Plasmodium target and mechanism of Torin 2 gametocytocidal activity using both directed and discovery approaches. In addition, to advancing our currently limited understanding of the cellular pathways required for gametocyte viability, the results will provide important new leads for the development of a potent malaria transmission-blocking drug.